CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway choice.

DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 (cell division cycle 23), a component of the E3-ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favouring error-prone NHEJ. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

Introduction: DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Methods: Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. Results: We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. Discussion: We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

Virtual Reality Rehabilitation Systems for Cancer Survivors: A Narrative Review of the Literature.

Rehabilitation plays a crucial role in cancer care, as the functioning of cancer survivors is frequently compromised by impairments that can result from the disease itself but also from the long-term sequelae of the treatment. Nevertheless, the current literature shows that only a minority of patients receive physical and/or cognitive rehabilitation. This lack of rehabilitative care is a consequence of many factors, one of which includes the transportation issues linked to disability that limit the patient's access to rehabilitation facilities. The recent COVID-19 pandemic has further shown the benefits of improving telemedicine and home-based rehabilitative interventions to facilitate the delivery of rehabilitation programs when attendance at healthcare facilities is an obstacle. In recent years, researchers have been investigating the benefits of the application of virtual reality to rehabilitation. Virtual reality is shown to improve adherence and training intensity through gamification, allow the replication of real-life scenarios, and stimulate patients in a multimodal manner. In our present work, we offer an overview of the present literature on virtual reality-implemented cancer rehabilitation. The existence of wide margins for technological development allows us to expect further improvements, but more randomized controlled trials are needed to confirm the hypothesis that VRR may improve adherence rates and facilitate telerehabilitation.

Cytotoxic evaluation and chemical investigation of tomatoes from plants (Solanum lycopersicum L.) grown in uncontaminated and experimentally contaminated soils.

The aim of this study was to evaluate the cytotoxic activity and the chemical composition of the tomato extracts coming from, Pomodoro Giallo and San Marzano Cirio 3, and then to evaluate the potential changes when plants were grown in soils contaminated by cadmium, chromium and lead. Extracts were investigated by UHPLC-HRMS and UV-Vis. Cell viability (CellTiter-Glo Luminescent assay), enzyme aldehyde dehydrogenase activity (ALDEFLOUR Assay), cell cycle progression (Accuri C6 Flow Cytometer), apoptosis and necrosis (Annexin V-FITC assay) were evaluated on two gastric cancer (AGS and NCI-N87) and two colorectal cancer (HT-29 and HCT 116) cell lines. Different content of polyphenol and carotenoid constituents was observed. Extracts from uncontaminated soil induced cytotoxic activity towards all selected cancer cells, while extracts coming from contaminated soils showed the aberrant phenotype increased in colorectal cancer cells. Chloroform extracts exerted the highest cytotoxic activity. AGS and HT-29 were the most sensitive to cell cycle arrest and to apoptosis. No necrotic effect was observed in HCT 116. The contrasting effects on cancer cells were observed based on tomato variety, the extract polarity, heavy metal identity, and tested cell line. The investigation of potential adverse health effects due to Cd in the fruits should be explored.

The Emerging Role of Cyclin-Dependent Kinase Inhibitors in Treating Diet-Induced Obesity: New Opportunities for Breast and Ovarian Cancers?

Overweight and obesity constitute the most impactful lifestyle-dependent risk factors for cancer and have been tightly linked to a higher number of tumor-related deaths nowadays. The excessive accumulation of energy can lead to an imbalance in the level of essential cellular biomolecules that may result in inflammation and cell-cycle dysregulation. Nutritional strategies and phytochemicals are gaining interest in the management of obesity-related cancers, with several ongoing and completed clinical studies that support their effectiveness. At the same time, cyclin-dependent kinases (CDKs) are becoming an important target in breast and ovarian cancer treatment, with various FDA-approved CDK4/6 inhibitors that have recently received more attention for their potential role in diet-induced obesity (DIO). Here we provide an overview of the most recent studies involving nutraceuticals and other dietary strategies affecting cell-cycle pathways, which might impact the management of breast and ovarian cancers, as well as the repurposing of already commercialized chemotherapeutic options to treat DIO.

Vanillin Prevents Doxorubicin-Induced Apoptosis and Oxidative Stress in Rat H9c2 Cardiomyocytes.

Doxorubicin (doxo) is an effective anticancer compound in several tumor types. However, as a consequence of oxidative stress induction and ROS overproduction, its high cardiotoxicity demands urgent attention. Vanillin possesses antioxidant, antiproliferative, antidepressant and anti-glycating properties. Therefore, we investigated the potential vanillin protective effects against doxo-induced cardiotoxicity in H9c2 cells. Using multiparametric approach, we demonstrated that vanillin restored both cell viability and damage in response to doxo exposure. Contextually, vanillin decreased sub-G1 appearance and caspase-3 and PARP1 activation, reducing the doxo-related apoptosis induction. From a mechanistic point of view, vanillin hindered doxo-induced ROS accumulation and impaired the ERK phosphorylation. Notably, besides the cardioprotective effects, vanillin did not counteract the doxo effectiveness in osteosarcoma cells. Taken together, our results suggest that vanillin ameliorates doxo-induced toxicity in H9c2 cells, opening new avenues for developing alternative therapeutic approaches to prevent the anthracycline-related cardiotoxicity and to improve the long-term outcome of antineoplastic treatment.

Targeted therapy based on p53 reactivation reduces both glioblastoma cell growth and resistance to temozolomide.

Glioblastoma (GB) is the most common and aggressive malignant tumor of the central nervous system. Despite current intensive treatment regimens, consisting of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy, the prognosis of patients with GB remains extremely poor. Considering that alterations of the p53 tumor suppressor pathway have a key role in both GB development and resistance to TMZ treatment, the re­activation of p53 could be an effective therapeutic approach against GB. In this study, we challenged p53 wild­type and mutant GB cell lines with RITA, a molecule originally identified for its ability to restore p53 functions, although it was subsequently shown to act also through p53­independent mechanisms. We examined the effects of RITA on GB cell viability, through MTS and clonogenic assays, and analyzed cell death through cytoflourimetric analyses. In all the tested GB cell lines, RITA significantly reduced the cell proliferative and clonogenic potential and induced cell accumulation in the S and/or G2/M cell cycle phases and massive p53­dependent apoptosis. Moreover, RITA was more effective than the well­known p53 re­activating molecule, nutlin­3, and did not affect the viability of normal astrocytes. In addition, RITA decreased survivin expression and induced DNA damage, two mechanisms that likely contribute to its anti­tumor effects. Furthermore, RITA synergized with TMZ and was able to decrease the expression of MGMT, which is a crucial player in TMZ resistance. Thus, although further studies are warranted to clarify the exact mechanisms of action of RITA, the data of this study suggest the potential of such an approach for GB therapy, which may also help to overcome resistance to TMZ.

A red orange and lemon by-products extract rich in anthocyanins inhibits the progression of diabetic nephropathy.

The major cause of end-stage renal disease is the diabetic nephropathy. Oxidative stress contributes to the development of type II diabetes mellitus (T2DM). In this study we have evaluated the effect of a diet with a new standardized of red orange and lemon extract (RLE) rich in anthocyanins (ANT) in the progression of the kidney disease on Zucker diabetic fatty rats. Oxidative stress and renal function were analyzed. In diabetic rats, the RLE restored the blood glucose levels, body weight, and normalized the reactive oxygen species (ROS) total pathways. The kidney inflammation, in diabetic rats, has not shown significant change, showing that the oxidative stress rather than to inflammatory processes is a triggering factor in the renal complication associated with T2DM. Therefore, the administration of the RLE prevents this complication and this effect could be related to the inhibition of ROS production.

24S-hydroxycholesterol affects redox homeostasis in human glial U-87 MG cells.

The cholesterol metabolite 24(S)-hydroxycholesterol (24S-OHC) allows cholesterol excretion from the brain and was suggested to be critically involved in physiological as well as neurodegenerative processes. It induces on human neuronal cell cultures a dose dependent toxicity associated with increased reactive oxygen species production. Since glial cells play a key role in assisting neuronal function, here we investigated the effects of increased concentrations of 24S-OHC on a glial cell model (human glioblastoma U-87 MG cells). We determined the content of PGC-1α and TFAM, involved in the biogenesis of mitochondria, both mitochondrial complexes activity and protein amount, lipid and protein oxidative damage, cellular reactive oxygen species (ROS) release and both the activities and amount of the antioxidant enzymes glutathione peroxidase and catalase. Low concentration of 24S-OHC increased cellular content of PGC-1α and TFAM and the activities of mitochondrial complexes I and II, with no marked changes in their protein amount. Interestingly, 24S-OHC at lower concentrations reduced while at higher concentration increased lipid and protein oxidative damage. Conversely, the content of nitro-tyrosine increased only with the highest 24S-OHC concentration. Also, cell H2O2 release was reduced by lower and increased by higher 24S-OHC used concentrations. The cell activity of glutathione peroxidase was reduced by 24S-OHC at higher concentration while that of catalase was reduced by all the assayed concentrations. Further, a dose dependent decrease of both enzymes levels was observed. In conclusion, we demonstrated that 24S-OHC exerts different effects on U-87 MG cells depending on its level. At lower concentrations it stimulates cellular processes critical to maintain redox homeostasis, while at higher dose its effect on the glial cell here used resemble its action on neurons.

Intrinsic structural versatility of the highly conserved 412-423 epitope of the Hepatitis C Virus E2 protein.

HCV infection is a major threaten for human health as it affects hundreds of million people worldwide. Here we investigated the conformational properties of the 412-423 fragment of the envelope E2 protein, one of the most immunogenic regions of the virus proteome whose characterization may provide interesting insights for anti-HCV vaccine development. The spectroscopic characterization of the polypeptide unravels its unexpected tendency to form amyloid-like aggregates. When kept in monomeric state, it shows a limited tendency to adopt regular secondary structure. Enhanced molecular dynamics simulations, starting from four distinct conformational states, highlight its structural versatility. Interestingly, all multiform conformational states of the polypeptide detected in crystallographic complexes with antibodies are present in the structural ensemble of all simulations. This observation corroborates the idea that known antibodies recognize this region through a conformational selection mechanism. Accordingly, the design of effective anti-HCV vaccines should consider the intrinsic flexibility of this region. The structural versatility of the 412-423 region is particularly puzzling if its remarkable sequence conservation is considered. It is likely that flexibility and sequence conservation are important features that endow this epitope with the ability to accomplish distinct functions such as immunity escape and interaction with host receptors.

RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines.

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

Virtual Reality as a Distraction Intervention to Relieve Pain and Distress During Medical Procedures: A Comprehensive Literature Review.

OBJECTIVES: This review aims to provide a framework for evaluating the utility of virtual reality (VR) as a distraction intervention to alleviate pain and distress during medical procedures. We first describe the theoretical bases underlying the VR analgesic and anxiolytic effects and define the main factors contributing to its efficacy, which largely emerged from studies on healthy volunteers. Then, we provide a comprehensive overview of the clinical trials using VR distraction during different medical procedures, such as burn injury treatments, chemotherapy, surgery, dental treatment, and other diagnostic and therapeutic procedures. METHODS: A broad literature search was performed using as main terms "virtual reality," "distraction," and "pain." No date limit was applied and all the retrieved studies on immersive VR distraction during medical procedures were selected. RESULTS: VR has proven to be effective in reducing procedural pain, as almost invariably observed even in patients subjected to extremely painful procedures, such as patients with burn injuries undergoing wound care, and physical therapy. Moreover, VR seemed to decrease cancer-related symptoms in different settings, including during chemotherapy. Only mild and infrequent side effects were observed. DISCUSSION: Despite these promising results, future long-term randomized controlled trials with larger sample sizes and evaluating not only self-report measures but also physiological variables are needed. Further studies are also required both to establish predictive factors to select patients who can benefit from VR distraction and to design hardware/software systems tailored to the specific needs of different patients and able to provide the greatest distraction at the lowest cost.

Antitumoral potential, antioxidant activity and carotenoid content of two Southern Italy tomato cultivars extracts: San Marzano and Corbarino.

Gastric cancer represents a diffuse and aggressive neoplasm, whose mortality index is among the highest in the world. Predisposing factors are E-cadherin mutations, Helicobacter pylori infection, and a diet rich in salted and smoked food, with a low intake of fresh fruits and vegetables. Here, we analyzed the effect of total lipophilic extracts of two Southern Italy tomato varieties, San Marzano and Corbarino, on an in vitro model of gastric cancer, YCC-1, YCC-2 and YCC-3 cell lines, characterized by different aggressiveness. Our results showed a possible role of these two varieties of tomatoes against typical neoplastic features. The treatment with tomato extracts affected cancer cell ability to grow both in adherence and in semisolid medium, reducing also cell migration ability. No toxic effects were observed on non-tumoral cells. We found, on gastric cancer cell lines, effects on both cell cycle progression and apoptosis modulation. The extent of antineoplastic effects, however, did not seem to correlate with the carotenoid content and antioxidant activity of the two tomato varieties. Our data indicate that San Marzano and Corbarino intake might be further considered as nutritional support not only in cancer prevention, but also for cancer patient diet.

Adrenaline induces mitochondrial biogenesis in rat liver.

We studied the effects of adrenaline administration and depletion (induced by reserpine) on rat liver oxidative metabolism. We showed that adrenaline increases, and reserpine decreases aerobic capacity (inferred by cytochrome oxidase activity) in tissue modifying the hepatic content of mitochondrial proteins without changing mitochondrial aerobic capacity. The changes in tissue cytochrome oxidase activity, which agreed with the expression levels of factors involved in mitochondrial biogenesis, such as PGC-1, NRF-1, and NRF-2, were associated with similar changes in tissue and mitochondrial State 3 respiration. Adrenaline and reserpine induced extensive lipid and protein oxidative damage in tissue and mitochondria. The increase in H2O2 release by respiring mitochondria and the decrease in the activities of the antioxidant enzymes glutathione peroxidase and reductase contributed to the reserpine effect on oxidative damage. The adrenaline effect is more difficult to explain, since the hormone increased the antioxidant enzyme activities but, in respiring mitochondria, increased ROS release rate in the presence of succinate and decreased it in the presence of pyruvate/malate. These opposite changes were due to the increased content of the autoxidizable electron carrier located at complex III and decreased content of that located at complex I. Our data suggest that adrenaline can be involved in the mitochondrial population adaptation which verify in conditions in which an increased body energy expenditure verify such as cold exposure.

NONO ubiquitination is mediated by FBW7 and GSK3 ß via a degron lost upon chromosomal rearrangement in cancer.

NONO is an RNA-binding protein involved in transcription, mRNA splicing, DNA repair, and checkpoint activation in response to UV radiation. NONO expression has been found altered in several tumor types, including prostate, colon, breast, melanoma, and in papillary renal carcinoma, in which an X chromosome inversion generates a NONO-TFE3 fusion protein. Upon such rearrangement, NONO loses its C-terminal domain. Through bioinformatics analysis, we identified a putative degron motif, known to be recognized by the Skp1-Cul1-F-box-protein (SCF) complex. Here, we evaluated how this domain could affect NONO protein biology. We showed that NONO interacts with the nuclear FBW7α isoform and its ubiquitination is regulated following modulation of the GSK3ß kinase. Mutation of T428A/T432A within the degron impaired polyubiquitination upon FBW7α and GSK3ß overexpression. Overall, our data suggest that NONO is likely subjected to proteasome-mediated degradation and add NONO to the list of proteins targeted by FBW7, which is itself often deregulated in cancer.

Structural Versatility of Hepatitis C Virus Proteins: Implications for the Design of Novel Anti-HCV Intervention Strategies.

BACKGROUND: HCV-linked pathologies represent worldwide health threats. Over the years, an enormous number of independent studies have been devoted to the understanding of the molecular bases of HCV infection. A significant amount of these investigations has been focused on the structural characterization of the virus proteins with the aim of developing structure-based innovative therapeutic approaches. An analysis of the current Protein Data Bank content unravels that the structural biology of the virus has hitherto covered a large fraction of the HCV proteins (75%). OBJECTIVE: The present review recapitulates the state-of-the-art of structural characterizations of HCV individual proteins with a specific focus on their structural versatility/flexibility. RESULTS: This survey indicates there is accumulating evidence that structural flexibility is a common feature among HCV proteins. This versatility can be detected at different structural level i.e. occurrence of alternative oligomeric states and/or of local and global flexibility. Somewhat surprisingly, some disordered or highly flexible regions of HCV proteins, such as the core and the antigenic fragment 412-423 of E2, present highly conserved sequences among the virus genotypes. The overall versatility of HCV proteins plays an important role in host protein recognition, drug resistance mechanisms, and virus escape from the host immunogenic system. Of particular relevance is the emerging idea that HCV uses local structural flexibility as an alternative tool to sequence variability to evade the immune response of the host organism. CONCLUSION: We believe that concepts emerged from this survey will be important for the development of anti-HCV vaccines that are eagerly needed.

The dynamic properties of the Hepatitis C Virus E2 envelope protein unraveled by molecular dynamics.

Hepatitis C Virus (HCV) is one of the most persistent human viruses. Although effective therapeutic approaches have been recently discovered, their use is limited by the elevated costs. Therefore, the development of alternative/complementary strategies is an urgent need. The E2 glycoprotein, the most immunogenic HCV protein, and its variants represent natural candidates to achieve this goal. Here we report an extensive molecular dynamics (MD) analysis of the intrinsic properties of E2. Our data provide interesting clues on the global and local intrinsic dynamic features of the protein. Present MD data clearly indicate that E2 combines a flexible structure with a network of covalent bonds. Moreover, the analysis of the two most important antigenic regions of the protein provides some interesting insights into their intrinsic structural and dynamic properties. Our data indicate that a fluctuating ß-hairpin represents a populated state by the region E2412-423. Interestingly, the analysis of the epitope E2427-446 conformation, that undergoes a remarkable rearrangement in the simulation, has significant similarities with the structure that the E2430-442 fragment adopts in complex with a neutralizing antibody. Present data also suggest that the strict conservation of Gly436 in E2 protein of different HCV genotypes is likely dictated by structural restraints. Moreover, the analysis of the E2412-423 flexibility provides insights into the mechanisms that some antibodies adopt to anchor Trp437 that is fully buried in E2. Finally, the present investigation suggests that MD simulations should systematically complement crystallographic studies on flexible proteins that are studied in combination with antibodies.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

UNLABELLED: The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ß-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE: Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a ß-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

"Cold training" affects rat liver responses to continuous cold exposure.

Continuous exposure of homeothermic animals to low environmental temperatures elicits physiological adaptations necessary for animal survival, which are associated to higher generation of pro-oxidants in thermogenic tissues. It is not known whether intermittent cold exposure (cold training) is able to affect tissue responses to continuous cold exposure. Therefore, we investigated whether rat liver responses to continuous cold exposure of 2 days are modified by cold training (1h daily for 5 days per week for 3 consecutive weeks). Continuous cold increased liver oxidative metabolism by increasing tissue content of mitochondrial proteins and mitochondrial aerobic capacity. Cold training did not affect such parameters, but attenuated or prevented the changes elicited by continuous cold exposure. Two-day cold exposure increased lipid hydroperoxide and protein-bound carbonyl levels in homogenates and mitochondria, whereas cold training decreased such effects although it decreased only homogenate protein damage in control rats. The activities of the antioxidant enzymes GPX and GR and H2O2 production were increased by continuous cold exposure. Despite the increase in GPX and GR activities, livers from cold-exposed rats showed increased susceptibility to in vitro oxidative challenge. Such cold effects were decreased by cold training, which in control rats reduced only H2O2 production and susceptibility to stress. The changes of PGC-1, NRF-1, and NRF-2 expression levels were consistent with those induced by cold exposure and cold training in mitochondrial protein content and antioxidant enzyme activities. However, the mechanisms by which cold training attenuates the effects of the continuous cold exposure remain to be elucidated.

KCTD5 is endowed with large, functionally relevant, interdomain motions.

The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed ß-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.